The goal of the proposed study Is to understand the long-term effects of angiotensin II (AII), renin-angiotensin system (ERAS) and other secretagogue! on the release of neurotransmitter and [Met5]enkephalin (ME) and the expression of the proenkephalin A (proENK) gene in bovine adrenal medullary (BAM) cells. the specific goal of this project was to elucidate the mechanisms for (1) the release of ME induced by AII, nicotine, TPA, PGE2, and arachidonic acid, and (2) the regulation of the expression of the proENK MRNA. Using primary cultured bovine adrenal chromaffin cells, we found that long-term stimulation with PGE2 and forskolin increased the release of renin from BAM cells. In addition to AII, angiotensinogen (a renin substrate) and angiotensin I also increased secretion of ME, which was reversed by captopril (an angiotensin converting enzyme inhibitor), indicating that ERAS plays an important role in long-term secretion of ME in BAM cells. We also studied the second messenger systems involved. Nicotine, angiotensin II, PMA and PGE2 increased both ME secretion and expression of the proENK MRNA. Calcium channel blockers (nitrendipine and nimodipine) effectively inhibited the responses induced by nicotine, AII and PGE,,, but not PMA. Calmidazolium (calmodulin antagonist) inhibited nicotine-, AII-., PGE2- but not PMA-induced responses. Furthermore, pertussis toxin (PTX), showed a synergistic effect for increasing ME secretion with nicotine, AII but not with PMA and PGE2- Recently, we assessed the role of PTX-sensitive G protein in FIE secretion. We found that PTX increased in a concentration dependent manner, suggesting the PTX-sensitive G protein may have tonic inhibitory effect in ME secretion. PTX-induced ME secretion was inhibited by nimodipine and calmidazolium. Forskolin also increased ME secretion and showed a synergistic effect, when combined with PTX. However, H7 (protein kinase C inhibitor) had no effect, suggesting that calcium calmodulin and cyclic AMP but not protein kinase C are major second messengers mediating PTX-induced responses.